The 5-methylated cytosine (5mC) turns off gene expression and the 5-hydroxymethylated cytosine (5hmC) turns it on. Both epigenetic modifications are thought to be one mechanism through which sociobehavioral factors regulate gene expression. However, the lab technique used in previous studies of DNA methylation related to sociobehavioral factors cannot distinguish 5mC from 5hmC and has other limitations. There is not any published study of 5hmC related to sociobehavioral factors. To date, the sociobehavioral determinants of DNA (hydroxy) methylation are largely unknown. Genetic factors can affect epigenetic modifications. Twins are genetically informative. Co-twins of a twin pair share germline genomic sequence [monozygotic twins (MZ) share 100% while dizygotic twins (DZ) on average share 50%)], germline epigenetic modifications inherited from parents, and common environment (i.e. environmental factors shared between co-twins, including age-cohort-period effects, family history of disease, maternal and other familial influences, sample processing and storage conditions). These shared factors can be uniquely controlled for in the co-twin study through comparing co-twins with each other, but not in a traditional epidemiologic study. In the NHLBI Twin Study, through physical examinations and in- person interviews, detailed data were collected on environmental factors from different aging stages, including sociobehavioral, psychological, dietary, lifestyle, biochemical, and clinical factors at exams 1 to 5 (1969-2000); data on vital status, cause of death, and age at death were collected through Dec 31, 2010; and buffy coat DNA samples were collected at age 60-74 years [exam 3 (1986-87)]. We have developed novel lab techniques that overcome the technical limitations mentioned above and can specifically measure both 5mC and 5hmC. Thus, by use of existing resources in the NHLBI Twin Study and the novel lab technique we have developed, we propose a co-twin study of the life-span sociobehavioral determinants of systemic DNA (hydroxy) methylation separated from genetic confounding. Our long-term research objective is to understand the interaction of sociobehavioral factors with the genome through epigenetic modifications and resultant disease consequences and the longevity, independent of the germline. In this R21 project, we will include a representative sample of 20 MZ and 20 DZ male twin pairs discordant for cardiovascular death and age at death from the NHLBI Twin Study of white male twins. These twins were born between 1917 and 1927, a period when 2 male MZ pairs and 4 male DZ pairs of twins were born per 1,000 live births. The specific aims of this R21 project are: 1) to measure genome-wide 5mC and 5hmC with our novel lab techniques, 2) to identify differentially 5-methylated genomic regions (DMRs) and 5- hydroxymethylated genomic regions (DhMRs), and 3) to explore sociobehavioral determinants of DMRs and DhMRs across the lifespan with life-span sociobehavioral data at exam 1. We aim to generate preliminary data for the future, large-scale study as stated in RFA-TW-13-002. The significance of our study lies in the development of sociobehavioral regimen to prolong the life expectancy.